The overall objective of this proposal is to elucidate the cellular and molecular mechanisms underlying the late phase of preconditioning (PC). Recent studies have shown that the generation of NO and the activation of the JAK-STAT pathway are necessary for the development of late PC, while iNOS and COX-2 act as obligatory mediators of protection. Our fundamental hypothesis is that the mechanism for the delayed antiapoptetic and infarct-sparing effects of both ischemia- and NO donor-induced late PC involves IL-6- dependent signaling via the JAK-STAT and ERK-C/EBP pathways which leads to upregulation of myocardial iNOS and COX-2. This study will utilize a broad multidisciplinary approach that will encompass the fields of integrative physiology, protein chemistry, molecular biology, and gene targeting. All studies will be carried out in an established mouse model of late PC. In Aim 1, the role of IL-6 in the genesis of ischemia-induced and NO donor-induced late PC will be conclusively verified using IL-6-/- mice. The effects of IL-6 on cardiomyocyte apoptosis as well as infarct size will be examined. The role of IL-6Ralpha in augmenting the protective effects of IL-6 will be determined in IL-6alpha overexpressing transgenic mice. Aim 2 will systematically investigate the activation of the JAK-STAT pathway by IL-6 and its role in the development of protection against apoptosis and infarction. Using STAT1-/- mice and a novel cardiac-specific STAT3-/- mouse, the role of specific STAT protein(s) will be conclusively identified. Aim 3 will examine IL-6- dependent ERK-C/EBP signaling in relation to the development of protection against apoptosis and infarction. The role of this pathway in both ischemic and NO donor PC will be conclusively established by the use of C/EBPbeta-/- mice. Aim 4 will elucidate the role of IL-6-activated JAK-STAT and ERK-C/EBP pathways in inducing myocardial iNOS 24 h later. Aim5 will determine the role of COX-2 induction via the IL-6-ERKC/ EBP axis in the mediation of delayed protection against apoptosis and infarction. COX-2-/- mice will be used to obtain conclusive results. This proposal will yield important new information regarding the antiapoptotic and delayed cardioprotective effects of IL-6 and its role in the induction of myocardial iNOS and COX-2, obligatory mediators of late PC. This information may eventually lead to the development of novel therapeutic strategies to protect the heart in patients with ischemic heart disease.